The present invention relates to an apparatus and method for rapidly disrupting cells or viruses using ultrasonic energy.
The extraction of nucleic acid from cells or viruses is a necessary task for many applications in the fields of molecular biology and biomedical diagnostics. Once released from the cells, the nucleic acid may be used for genetic analysis, e.g., sequencing, pathogen identification and quantification, nucleic acid mutation analysis, genome analysis, gene expression studies, pharmacological monitoring, storing of DNA libraries for drug discovery, etc. The genetic analysis typically involves nucleic acid amplification and detection using known techniques. For example, known polynucleotide amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR), QB replicase amplification (QBR), self-sustained sequence replication (3SR), strand-displacement amplification (SDA), xe2x80x9cbranched chainxe2x80x9d DNA amplification, ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), and cycling probe reaction (CPR).
The extraction of nucleic acids from cells or viruses is generally performed by physical or chemical methods. Chemical methods typically employ lysing agents. (e.g., detergents, enzymes, or strong organics) to disrupt the cells and release the nucleic acid, followed by treatment of the extract with chaotropic salts to denature any contaminating or potentially interfering proteins. Such chemical methods are described in U.S. Pat. No. 5,652,141 to Henco et al. and U.S. Pat. No. 5,856,174 to Lipshutz et al. One disadvantage to the use of harsh chemicals for disrupting cells is that the chemicals are inhibitory to subsequent amplification of the nucleic acid. In using chemical disruption methods, therefore, it is typically necessary to purify the nucleic acid released from the cells before proceeding with further analysis. Such purification steps are time consuming, expensive, and reduce the amount of nucleic acid recovered for analysis.
Physical methods for disrupting cells often do not require harsh chemicals that are inhibitory to nucleic acid amplification (e.g., PCR). These physical methods, however, also have their disadvantages. For example, one physical method for disrupting cells involves placing the cells in a solution and heating the solution to a boil to break open the cell walls. Unfortunately, the heat will often denature proteins and cause the proteins to stick to the released nucleic acid. The proteins then interfere with subsequent attempts to amplify the nucleic acid. Another physical method is freeze thawing in which the cells are repeatedly frozen and thawed until the cells walls are broken. Unfortunately, freeze thawing often fails to break open many structures, most notably certain spores and viruses that have extremely tough outer layers.
Another physical method for disrupting cells is the use of a pressure instrument. With this method, a solution of mycobacterial microorganisms is passed through a very small diameter hole under high pressure. During passage through the hole, the mycobacteria are broken open by the mechanical forces and their internal contents are spilled into solution. Such a system, however, is large, expensive and requires a cooling system to prevent excessive heat from building up and damaging the contents of the lysed cells. Moreover, the instrument needs to be cleaned and decontaminated between runs and a large containment system is required when infectious material is handled. A further disadvantage to this system is that the solution must contain only particles having substantially the same size, so that it may not be used to process many untreated clinical or biological specimens.
It is also known that cells can be lysed by subjecting the cells to ultrasonic agitation. This method is disclosed by Murphy et al. in U.S. Pat. No. 5,374,522. According to the method, solutions or suspensions of cells are placed in a container with small beads. The container is then placed in an ultrasound bath until the cells disrupt, releasing their cellular components. This method has several disadvantages. First, the distribution of ultrasonic energy in the bath is not uniform, so that a technician must locate a high energy area within the bath and place the container into that area. The non-uniform distribution of ultrasonic energy also produces inconsistent results. Second, the ultrasound bath does not focus energy into the container so that the disruption of the cells often takes several minutes to complete, a relatively long period of time when compared to the method of the present invention. Third, it is not practical to carry an ultrasound bath into the field for use in biowarfare detection, forensic analysis, or on-site testing of environmental samples.
The present invention overcomes the disadvantages of the prior art by providing an improved apparatus and method for disrupting cells or viruses to release the nucleic acid therefrom. In contrast to the prior art methods described above, the present invention provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals. The disruption of the cells or viruses can often be completed in 5 to 10 seconds. In addition, the apparatus and method of the present invention provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next.
The apparatus includes a container having a chamber for holding the cells or viruses. The container has at least one wall defining the chamber. The apparatus also includes an ultrasonic transducer, preferably an ultrasonic horn, for contacting an external surface of the wall and for transmitting ultrasonic energy into the chamber through the wall. The apparatus further includes a support structure for holding the container and the transducer against each other such that the transducer contacts the external surface of the wall and for applying to the container or to the transducer a substantially constant force to press together the transducer and the wall. In a preferred embodiment, the support structure comprises a base, a first holder attached to the base for holding the transducer, and a second holder for holding the container. The second holder is slidably mounted to the base for positioning the container against the transducer such that the external surface of the wall contacts the transducer. The support structure also includes at least one elastic body (e.g., a spring) for applying to the second holder the substantially constant force to press the wall against the transducer.
In operation, a liquid or gel containing the cells or viruses is held in the chamber of the container. Beads are also preferably placed in the chamber to enhance the disruption of the cells or viruses. The ultrasonic transducer is then held against the wall of the chamber, and the constant force is applied to the container or to the transducer to press together the transducer and the wall. Ultrasonic energy is then transmitted from the transducer into the chamber to disrupt the cells or viruses, releasing the nucleic acid therefrom. The apparatus and method are effective for rapidly and consistently lysing cells or viruses without the use of harsh chemicals that inhibit nucleic acid amplification.